CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

. Architecture of TMs 3, 10 and 11 in bovine OF (yellow) and IF (salmon) conformation illustrating the ~5 Å vertical shift of the ion coordination site between these conformations (evaluated as center of mass shift for the displayed residues). The Cα atoms of the core residues from the central binding site S1 5 are shown as spheres and labeled accordingly.

Supplementary Fig. 12. RMSD plots for the 1 µs MD simulations of bAE1 dimers.
Relevant structural changes during the simulations (i.e. bending of TM11 and occlusion of the IF cavity by motion of TM10) that impact the RMSD are marked with an asterisk. The first 50 ns of the trajectories were discarded from the RMSD analysis.

Coarse-Grained Metadynamics Simulations
We employed coarse-grained metadynamics simulations to probe the free energy landscape of the IF to OF conformational transition and to obtain a qualitative understanding about which state is more energetically favorable. Three collective variables (CVs) were employed for the description of the IF-OF transition. The CVs were selected using the immobile gate (TM 5-7, 12-14) part as the reference. The center of mass (COM) distances of the core helices (TM 1-4, [8][9][10][11] from the COM of the gate was calculated for both the IF and OF states. The COM distances of these helices from the core part were used as CVs if they varied more than 2 Å, which led to three CVs, namely COM distances between TM3-gate, TM10-gate and TM11-gate.
The simulations were performed with the Martini 3.0 force field 8  we performed the metadynamics calculations for 10 µs in NPT ensembles using the same timestep and integrator. The system pressure and temperature were set to 1 bar and 310 K, respectively; the pressure was maintained using Parrinello-Rahman barostat and the temperature was maintained using v-rescale thermostat. A semi-isotropic pressure coupling was used to maintain the shape of the bilayer where bilayer lateral dimensions were coupled. Pair list was generated using the Verlet scheme for the nonbonded interactions. The coulombic terms were calculated using reaction-field electrostatics with a cut-off of 1.1 nm and the relative dielectric constant was set to 15. A cutoff value of 1.1 nm was used for the VdW terms using a potentialshift with Verlet cutoff-scheme.
The metadynamics calculations made use of the three collective variables mentioned above. The height of the gaussian was chosen as 0.5 kJ/mol, and sigma (width) was set to 0.05. The gaussians were deposited every 500 timesteps.
Due to the coarse-grained nature of the metadynamics protocol, the complexity of the assessed conformational transition, the lack of bound substrates, and the presence of only one monomer in the simulation, the intrinsic convergence of the metadynamics results is not suitable for assessment of their accuracy. Thus, our coarse-grained metadynamics results should be viewed from a qualitative (e.g. which state is of lower energy) rather than a quantitative (exact energy differences) point of view. All-atom metadynamics simulations which address the deficiencies mentioned above and can provide quantitatively accurate energetics for the IF to OF transition are currently underway.